A method for the in vitro evolution of nucleic acid molecules with highly specific binding to target molecules has been developed. This method, Systematic Evolution of Ligands by EXponential enrichment, termed the SELEX combinatorial chemistry process, is described in U.S. patent application Ser. No. 07/536,428, entitled "Systematic Evolution of Ligands by Exponential Enrichment", now abandoned, U.S. patent application Ser. No. 07/714,131, filed Jun. 10, 1991, entitled "Nucleic Acid Ligands", now U.S. Pat. No. 5,475,096, U.S. patent application Ser. No. 07/931,473, filed Aug. 17, 1992, entitled "Methods For Identifying Nucleic Acid Ligands", now U.S. Pat. No. 5,270,163 (see also WO91/19813), each of which is herein specifically incorporated by reference. Each of these applications, collectively referred to herein as the SELEX Patent Applications, describes a fundamentally novel method for making a nucleic acid ligand to any desired target molecule.
The SELEX method involves selection from a mixture of candidate oligonucleotides and step-wise iterations of binding, partitioning and amplification, using the same general selection scheme, to achieve virtually any desired criterion of binding affinity and selectivity. Starting from a mixture of nucleic acids, preferably comprising a segment of randomized sequence, the SELEX method includes steps of contacting the mixture with the target under conditions favorable for binding, partitioning unbound nucleic acids from those nucleic acids which have bound specifically to target molecules, dissociating the nucleic acid-target complexes, amplifying the nucleic acids dissociated from the nucleic acid-target complexes to yield a ligand-enriched mixture of nucleic acids, then reiterating the steps of binding, partitioning, dissociating and amplifying through as many cycles as desired to yield highly specific, high affinity nucleic acid ligands to the target molecule.
The basic SELEX method has been modified to achieve a number of specific objectives. For example, U.S. patent application Ser. No. 07/960,093, filed Oct. 14, 1992, entitled "Method for Selecting Nucleic Acids on the Basis of Structure", abandoned in favor of U.S. patent application Ser. No. 08/198,670, now U.S. Pat. No. 5,707,796, describes the use of the SELEX process in conjunction with gel electrophoresis to select nucleic acid molecules with specific structural characteristics, such as bent DNA. U.S. patent application Ser. No. 08/123,935, filed Sep. 17, 1993, entitled "Photoselection of Nucleic Acid Ligands" describes a SELEX-based method for selecting nucleic acid ligands containing photoreactive groups capable of binding and/or photocrosslinking to and/or photoinactivating a target molecule. U.S. patent application Ser. No. 08/134,028, filed Oct. 7, 1993, entitled "High-Affinity Nucleic Acid Ligands That Discriminate Between Theophylline and Caffeine", abandoned in favor of U.S. patent application Ser. No. 08/443,957, now U.S. Pat. No. 5,580,737, describes a method for identifying highly specific nucleic acid ligands able to discriminate between closely related molecules, termed Counter-SELEX. U.S. patent application Ser. No. 08/143,564, filed Oct. 25, 1993, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Solution SELEX", abandoned in favor of U.S. patent application Ser. No. 08/461,069, now U.S. Pat. No. 5,567,588, describes a SELEX-based method which achieves highly efficient partitioning between oligonucleotides having high and low affinity for a target molecule. U.S. patent application Ser. No. 07/964,624, filed Oct. 21, 1992, entitled "Nucleic Acid Ligands to HIV-RT and HIV-1 REV" now U.S. Pat. No. 5,496,938 describes methods for obtaining improved nucleic acid ligands after the SELEX process has been performed. U.S. patent application Ser. No. 08/400,440, filed Mar. 8, 1995, entitled "Systematic Evolution of Ligands by EXponential Enrichment: Chemi-SELEX", now U.S. Pat. No. 5,705,337, describes methods for covalently linking a ligand to its target.
The SELEX method encompasses the identification of high-affinity nucleic acid ligands containing modified nucleotides conferring improved characteristics on the ligand, such as improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitutions at the ribose and/or phosphate and/or base positions. SELEX-identified nucleic acid ligands containing modified nucleotides are described in U.S. patent application Ser. No. 08/117,991, filed Sep. 8, 1993, entitled "High Affinity Nucleic Acid Ligands Containing Modified Nucleotides", abandoned in favor of U.S. patent application Ser. No. 08/430,709, now U.S. Pat. No. 5,660,985, that describes oligonucleotides containing nucleotide derivatives chemically modified at the 5- and 2'-positions of pyrimidines. U.S. patent application Ser. No. 08/134,028, supra, describes highly specific nucleic acid ligands containing one or more nucleotides modified with 2'-amino (2'-NH.sub.2), 2'-fluoro (2'-F), and/or 2'-O-methyl (2'-OMe). U.S. patent application Ser. No. 08/264,029, filed Jun. 22, 1994, entitled "Novel Method of Preparation of Known and Novel 2' Modified Nucleosides by Intramolecular Nucleophilic Displacement", describes methods for making various 2'-modified nucleosides.
The SELEX method encompasses combining selected oligonucleotides with other selected oligonucleotides and non-oligonucleotide functional units as described in U.S. patent application Ser. No. 08/284,063, filed Aug. 2, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Chimeric SELEX", now U.S. Pat. No. 5,637,459, and U.S. patent application Ser. No. 08/234,997, filed Apr. 28, 1994, entitled "Systematic Evolution of Ligands by Exponential Enrichment: Blended SELEX", now U.S. Pat. No. 5,683,867, respectively. These applications allow the combination of the broad array of shapes and other properties, and the efficient amplification and replication properties, of oligonucleotides with the desirable properties of other molecules. Each of the above described patent applications which describe variations of the basic SELEX procedure are specifically incorporated by reference herein in their entirety.
Without question, the SELEX process is very powerful. The nucleic acid ligands obtained by the SELEX process have the ability to act in many capacities. One of the capacities that nucleic acid ligands possess is the ability to bind specifically to a target compound.
Ligands derived by the SELEX process have been used in other diagnostic applications, including in U.S. patent application Ser. No. 08/487,425, filed Jun. 7, 1995, entitled "Enzyme Linked Oligonucleotide Assays ELONAS" and U.S. patent application Ser. No. 08/479,729, filed Jun. 7, 1995, entitled "Use of Nucleic Acid Ligands in Flow Cytometry", both of which are herein incorporated by reference in their entirety.
Specific and high affinity molecular recognition is critical for diagnostic applications. Until recently, engineering of molecules that recognize targets has been mainly limited to proteins. Protein molecules that recognize a specific target have typically been generated as antibodies. As a result, antibodies have received a central role in the development of analytical and separation methods that are currently employed. The methods which primarily use antibodies include, immunometric assays, such as enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays, flow cytometry diagnostics, blotting applications, fluorescent anisotropy, membrane assays, biosensors, etc.
Blotting applications are currently being used as confirmatory diagnostics tests for various disease states, including HIV and Hepatitis C. Blotting applications are also frequently used in the research laboratory.
Immunometric assays have been found to be particularly well suited for the detection of polyvalent targets or antigens, i.e., antigenic substances that are able to complex with two or more antibodies at the same time. Such assays typically employ a quantity of unlabelled antibody bound to a solid support that is insoluble in the fluid being tested and a quantity of soluble antibody bearing a label such as an enzyme or a radioactive isotope that permits detection and/or a quantitative estimate of the ternary complex formed between solid phase antibody, antigen and labeled antibody. Details regarding immunometric assays are provided in U.S. Pat. No. 4,486,530.
Immunoblots have recently been used as diagnostic and research tools. A major advance in the analysis of protein-nucleic acid interactions occurred in 1980, when Bowen et al. (Nucl. Acids Res. (1980) 8:1-20) established a method to detect protein-DNA interactions by binding labeled DNA to proteins which had been separated by SDS-PAGE and transferred to nitrocellulose ("Southwestern blotting"). This procedure was extended to protein-RNA binding ("Northwestern blotting") by demonstrating protein binding to .sup.125 I-labeled Rous sarcoma virus RNA. Most applications of Northwestern blotting have been performed on purified proteins and RNA. There are few examples of the application of Northwestern blotting to detect interactions between specific proteins and mature mRNAs, even though these interactions are important in the translational control of gene expression. There are even fewer examples of the use of the Northwestern blotting method to detect generalized protein-mRNA interactions. One reason for this is that the Northwestern blotting procedure is difficult to apply in a more generalized manner, as the application of the method to search for RNA-binding proteins in the context of a population of hundreds or thousands of proteins is usually confounded by high backgrounds and lack of reproducibility.
An immunoblot is a sensitive immunoassay method for determining whether a substance contains a certain target. Usually an antigen is detected using an antibody specific for the antigen by means of a detection enzyme chemically coupled to the antibody or some other species that has a detectable property such as radioactivity, fluorescence, etc. Typically, a substance which may or may not contain the target compound of interest is attached to a solid support. The matrix-bound substance is contacted with an antibody. After washing, a detection system indicates whether an interaction between the substance and the antibody has occurred. The detection system can be any detection system known to one of ordinary skill in the art, and can include, but is not limited to, enzyme-linked nucleic acid ligands, radiolabeled nucleic acid ligands, enzyme-linked secondary antibody or universal antibody (Protein A), and chemiluminescence detection systems.
In addition to antibodies, oligonucleotides are also being used in diagnostics, but in a different manner. Sequence information of oligonucleotide probes is used to specifically target genomic complementary base sequences in techniques such as Southern blotting, in situ hybridization and polymerase chain reaction (PCR)-based amplifications. However, in these processes information stored in an oligonucleotide is only generally used to detect nucleic acid molecules, and naturally occurring nucleic acid binding proteins. The information content (linear sequence) of nucleic acids predominantly relies on Watson/Crick base pairing and can only discriminate among DNAs and RNAs or the sequence specific nucleic acid binding proteins.
Currently, oligonucleotides are being used in a western blotting format (Northwestern blots) only for the detection of naturally occurring oligonucleotide binding proteins. Specific oligonucleotide probes are used to specifically detect sequence specific or non-sequence dependent nucleic acid binding proteins (Chen, et al., BBRC (1993) 191:18-25).
Diagnostic nucleic acids known to date and most antibodies are known to recognize linear epitopes within a nucleic acid or protein, respectively. Most antibodies are known to recognize linear epitopes within a protein, presumably due to the presentation of peptide fragments by antigen-presenting cells. However, in the SELEX process an intact protein is repeatedly presented to pools of oligonucleotides increasingly enriched in oligonucleotides having an affinity to the intact protein. Hence, SELEX-derived oligonucleotide ligands tend to recognize conformational epitopes. Relying on structural content (three-dimensional structures), nucleic acid ligands can be used in diagnostic applications for any type of target. Before the SELEX process, the structural content of nucleic acids was essentially not appreciated and there was no way to utilize the structural capabilities of nucleic acids in diagnostics.
The use of nucleic acid ligands in blot-type diagnostic assays, which were previously believed to depend on antibody recognition, has not been demonstrated thus far. Ligand binding information stored in the three dimensional structure of an oligonucleotide is useful for the detection of target molecules in substances, including protein targets that do not normally function to bind nucleic acids. The present invention demonstrates that oligonucleotides that bind with high affinity to their cognate target compounds can replace antibodies in a matrix-bound target detection format. More specifically, SELEX-derived oligonucleotides that bind with high affinity to human VEGF, hCG and hTSH can replace antibodies in a western blot format.